Methods and Devices for the Treatment of Food Allergies

ABSTRACT

Methods and devices are provided for treating a food allergy in a subject in need thereof. The method entails delivering an effective amount of an allergen associated with the food allergy into the subject&#39;s cutis skin layer. Delivering the allergen is carried out by inserting one or more allergen-coated solid microneedles into the subject&#39;s skin. The one or more solid microneedles each has a base, shaft and tip, and when inserted in the subject, do not extend beyond the cutis. The allergen is allowed to dissociate from the one or more microneedles while inserted in the subject&#39;s cutis. Once the allergen disassociates, the one or more microneedles is removed from the subject&#39;s skin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/616,171, filed on Nov. 22, 2019, which is the National Stage ofInternational Application No. PCT/US2018/035399, filed on May 31, 2019,which claims priority to U.S. Provisional Patent Application Ser. No.62/512,884 filed on May 31, 2017 and U.S. Provisional Patent ApplicationSer. No. 62/576,176 filed on Oct. 24, 2017. The contents of allapplications are incorporated by reference herein in their entirety.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number1R01AI121322-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods, devices and composition forthe treatment of food allergies.

INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

The present application includes a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 30, 2018, isnamed TECH2103WO_SeqList and is 6 kilobytes in size.

BACKGROUND OF THE INVENTION

Peanut allergy is a life-threatening condition. About 1% of the U.S.population (˜3 million people) has peanut allergies, and there is noFDA-approved treatment. Strict avoidance, and a peanut-free diet is theonly option available to manage peanut allergies, which imposes severelimitations on the lifestyle of the patient and their families. Patientsare also advised to carry an epinephrine injection to mitigateanaphylaxis, which can occur due to accidental peanut exposure.Adherence to a peanut-free diet imposes severe limitations on thelifestyle of the patient and their families, and reduces their qualityof living. Importantly, restricted food diets in children can lead tonutritional deficiencies, and as such, methods for the treatment ofpeanut allergies are of great interest.

Peanut allergy is associated with an abnormal immune response to peanutproteins and it is mediated by peanut specific IgE antibodies. Allergicreaction to peanut in food can produce diverse symptoms including skinrashes, gastrointestinal reactions such as pain and vomiting, and even asevere life-threatening anaphylactic reaction (Sampson et al. (2014 JAllergy Clin Immunol. 134(5), pp. 1016-1025 e43). Oral immune-therapy(OIT) for peanut allergies is relatively new and experimental, and itaims to modulate this aberrant IgE response. The first open-label trialfor peanut OIT was published in 2009 (Hofmann et al. (2009). J AllergyClin Immunol. 124(2), pp. 286-291, 291.e1-6; Jones et al. (2009). JAllergy Clin Immunol. 124(2), pp. 292-300.e97). The published protocolsfor peanut OIT (and food OIT in general) typically involve oral deliveryof peanut flour/protein or extract in: (i) a rapid/rush dose escalationphase lasting one day (peanut protein dose increased from about 100 μgto 50 mg), (ii) a gradual dose buildup phase lasting many months (peanutprotein dose increased to hundreds and thousands of milligram), and(iii) maintenance phase lasting months to years (peanut protein dosemaintained at several thousand milligram). (Wood (2016). J Allergy ClinImmunol. 137(4), pp. 973-982; Deol and Bird (2014). Hum Vaccin.Immunother. 10(10), pp. 3017-21; Sampson (2013). J Allergy ClinImmunol.: In Practice 1(1), pp. 15-21.

The end-point of most peanut OITs has been to reorient the abnormalimmune response and to desensitize the patient to peanut (Deol and Bird(2014). Hum Vaccin. Immunother. 10(10), pp. 3017-21; Sampson (2013). JAllergy Clin Immunol.: In Practice 1(1), pp. 15-21; Commins et al.(2016). Current Allergy and Asthma Reports 16(5), p. 35; Wood (2017). JInvestig Allergol Clin Immunol, p. 0). Desensitization means increasingthe patient's threshold to peanut reactivity (i.e., the amount of peanutthat can be safely tolerated by the patient). To maintaindesensitization, the patient is required to continue ingesting peanutsat a maintenance dose at regular intervals. However, sustainedunresponsiveness, i.e., the ability of the patient to be non-responsiveto peanut ingestion after completion of OIT without the need to be on amaintenance dose, is the desirable treatment endpoint. In 2011 the firstdata of sustained unresponsiveness was published, and it was shown that50% of the subjects were unresponsive to peanut 4 weeks after OIT(Vickery et al. (2014). J Allergy Clin Immunol. 133(2), pp. 468-475.e6).In another study, it was found that 7/20 subjects were unresponsive 3months after stopping OIT, and only 3 out of these 7 were unresponsiveanother 3 months later (i.e., 3/20 were unresponsive 6 months afterstopping OIT) (Syed et al. (2014). J Allergy Clin Immunol. 133(2): pp.500-510.el 1).

Current peanut OIT protocols require daily ingestion of peanut, and thedose is continuously increased to thousands of milligrams of peanutprotein. Adverse events such as abdominal pain, vomiting, upperrespiratory reactions (sneezing and congestion), and skin rashes/hivesare very common, especially during the initial rush dose escalation frommicrograms to tens of milligram in a single day (Hofmann et al. (2009).J Allergy Clin Immunol. 124(2), pp. 286-91, 291.e1-6), and the dosebuildup phase when the peanut dose is raised from tens to thousands ofmilligrams (Vickery et al. (2017). J Allergy Clin Immunol. 139(1), pp.173-181 e8). In one study, a direct correlation was observed betweenasthma and peanut OIT, wherein it was found that asthmatic patientsexperienced respiratory adverse events (Hofmann et al. (2009). J AllergyClin Immunol 124(2), pp. 286-91, 291.e1-6.).

Allergy shots, which are subcutaneously delivered, have a proven trackrecord to provide long term treatment for environmental allergens thatcause respiratory symptoms such as allergic rhinitis, allergicconjunctivitis, allergic asthma, or insect allergy (including bee venom)(Cox et al. (2011). The Journal of allergy and clinical immunology 127(1Suppl), pp. S1-55). Building on this success, peanut subcutaneousimmunotherapy was attempted in 1990s (Oppenheimer et al. (1992). JAllergy Clin Immunol 90(2), pp. 256-62; Nelson et al. (1997). J AllergyClin Immunol. 99(6 Pt 1), pp. 744-51). Patients underwent rushimmunotherapy, whereby they received four injections per day ofincreasing doses of peanut extract for five consecutive days, and thenreceived eight injections (1/week) of maintenance dose. This was a smallclinical study, and in three of the six patients, 67-100% reduction insymptoms was seen when they were orally challenged with peanut (Nelsonet al. (1997). J Allergy Clin Immunol. 99(6 Pt 1), pp. 744-51). However,the systemic reactions were high. During rush immunotherapy, 23% of theinjections given to the patients led to a systemic reaction needingepinephrine injection, while during maintenance phase this reaction ratewas 33%. Based in part on these high reaction rates, the studies werehalted. Subcutaneous injections have not been attempted again.

To circumvent the high reaction rate seen when peanut allergen isinjected subcutaneously, a skin patch containing peanut allergen (100,250 or 500 kg) was developed (34-38). This patch has now also completeda clinical trial (Jones et al. (2017). J Allergy Clin Immunol. 139(4),pp. 1242-1252), for which the patch was applied to the skin continuouslyfor 24 hr. After 24 hr., the patch was removed and a new patch wasimmediately placed on a different skin site. Thus, the patient receiveda skin patch continuously, every day for one year. Low reaction rateswere seen, and mostly these were observed topically on the skin. None ofthe patients could successfully complete the oral food challenge of 1044mg (Jones et al. (2016). Consortium of Food Allergy, Epicutaneousimmunotherapy for the treatment of peanut allergy in children and youngadults. J Allergy Clin Immunol). In contrast, peanut oral immunotherapy,although associated with a higher number of adverse events, has allowedpatients to successfully complete oral challenges with about 4000 mgpeanut protein (Hofmann et al. (2009). J. Allergy Clin. Immunol. 124(2),pp. 286-91, 291.e1-6; Jones et al. (2009). J. Allergy Clin. Immunol.124(2), pp. 292-300.e97; Blumchen et al. (2010). J. Allergy Clin.Immunol. 126(1), pp. 83-91).

Based on the results of these clinical studies, new treatment optionsfor peanut and other food allergies would be beneficial and an advancein the field. The present invention addresses this and other needs.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method fortreating a food allergy in a subject in need thereof. In one embodiment,the method for treating the food allergy comprises delivering aneffective amount of an allergen associated with the food allergy intothe subject's cutis. The delivering step comprises inserting one or moresolid microneedles each having a base, shaft and tip into the subject'scutis. At least one of the one or more solid microneedles is coated withallergen associated with the food allergy and the one or moremicroneedles do not extend beyond the cutis once inserted. The allergenis allowed to dissociate from the microneedles while inserted in thesubject's cutis. Once the allergen dissociates, the one or moremicroneedles are removed from the subject's skin.

The subject in one embodiment is a human. In a further embodiment, thehuman subject is from about 2 to about 12 years old, e.g., from about 4to about 12 years old or from about 4 to about 10 years old. In oneembodiment, the one or more solid microneedles comprise a microneedlearray of two or more solid microneedles, for example, from about 10 toabout 100 solid microneedles.

In one embodiment of the method, the one or more solid microneedlesextends from an adhesive substrate. In one embodiment, the one or moresolid microneedles are stainless steel. In a further embodiment, theaverage length of the one or more solid microneedles is from about 100μm to about 1000 μm, as measured from the base of the tip. In even afurther embodiment, the average length of the one or more solidmicroneedles is from about 200 μm to about 900 μm.

The one or more solid microneedles used in the methods and devicesprovided herein, in one embodiment, comprises from about 10 to about 150microneedles, for example, from about 10 to about 100 microneedles, fromabout 10 to about 80 microneedles, or from about 20 to about 70microneedles.

The methods provided herein are not limited to the type of food allergythat is treatable. For example, in one embodiment, the food allergy is agroundnut, peanut, milk, egg, tree nut, seed, fish, shellfish,crustacean, cereal, legume allergy, or a combination thereof. In oneembodiment, the food allergy is a peanut allergy. In embodiments where apeanut allergy is treated, the peanut allergen can comprise Ara h1, Arah2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Arah11, Ara h12, Ara h13, a peptide fragment thereof, or a combinationthereof. In a further embodiment, the peanut allergen comprises Ara h1,Ara h2, Ara h6, a peptide fragment thereof, or a combination thereof.Additionally, where a peanut allergen is coated on the microneedlearray, it can be provided as peanut protein extract, protein flour, or acombination thereof.

In one embodiment of the method, once inserted into the cutis, thetip(s) of the one or more microneedles do not extend beyond theepidermis skin layer. In another embodiment, once inserted into thecutis, the tip(s) of the one or more microneedles do not extend beyondthe dermis skin layer.

As provided above, in one embodiment, a method for treating a foodallergy is provided comprising in part, inserting one or more solidmicroneedles into the subject's cutis, wherein at least one microneedleof the one or more solid microneedles is coated with allergen associatedwith the food allergy, and the at least one coated microneedle of thearray does not extend beyond the cutis once inserted. In a furtherembodiment, each microneedle of the one or more microneedles does notextend beyond the cutis once inserted. The allergen is allowed todissociate from the at least one microneedle while inserted in thesubject's cutis. In a further embodiment, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or at least about 90% of the allergen disassociates from the at leastone microneedle while inserted in the subject's cutis. Disassociation ofthe allergen in one embodiment, is carried out for about 1 minute toabout 10 minutes, for example from about 1 minute to about 6 minutes orfrom about 1 minute to about 5 minutes.

As provided above, in one embodiment, a subject is treated for foodallergy via delivering an effective amount of an allergen associatedwith the food allergy into the subject's cutis. Delivering is carriedout via inserting one or more solid microneedles into the subject'scutis, wherein the one or more solid microneedles each comprises a base,shaft and tip. At least one microneedle of the one or more solidmicroneedles is coated with allergen associated with the food allergyand the at least one coated microneedle of the array does not extendbeyond the cutis once inserted. In a further embodiment, eachmicroneedle of the one or more solid microneedles does not extend beyondthe cutis once inserted. The allergen is allowed to dissociate from theat least one microneedle while inserted in the subject's cutis. Once theallergen dissociates, the one or more solid microneedles is removed fromthe subject's skin. This method in a further embodiment, is carried outserially, over time. For example, in one embodiment, the method iscarried out once daily, twice daily, three times daily, every other day,twice a week, or once weekly. In a further embodiment, the delivering aneffective amount of an allergen results in delivering substantially thesame amount of allergen each time the method is carried out. In anotherembodiment, the delivering an effective amount of an allergen results indelivering an escalating dosage of the allergen at least once, at leasttwice, or at least three times.

In one embodiment of the method of treating food allergy, the treatingresults in desensitization to the allergen. For example, in oneembodiment, desensitization is by at least about 2%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, about 70%, about 75%, at least about 80%,at least about 85%, or at least about 90% as compared to the subjectprior to commencing the treatment, a subject receiving a placebo or asubject not receiving the treatment.

In one embodiment of the method of treating food allergy, the treatingresults in a decrease in the number of allergen specific IgE antibodiesin the subject, as compared to the number secreted prior to thetreating. In another embodiment, the treating results in an increase inthe number of allergen specific IgG antibodies in the subject, ascompared to the number secreted prior to the treating. In a furtherembodiment, the subject is human and the allergen specific IgGantibodies are allergen specific IgG4 antibodies. In another embodiment,the treating results in a decreased number of mast cells in the subject,as compared to the number secreted prior to the treating. In a furtherembodiment, the decreased number of mast cells is at the site ofallergen exposure (e.g., the GI tract for food allergens) in thesubject, as compared to the number secreted at the site prior to themethod being carried out. In yet another embodiment, the treatingresults in a decreased number of basophils in the subject, as comparedto the number secreted prior to the treating. In a further embodiment,the decreased number of basophils is at the site of allergen exposure(e.g., the GI tract for food allergens) in the subject, as compared tothe number secreted at the site prior to the method being carried out.In even another embodiment, the treating results in an increasedcytokine production in the subject, as compared to the number secretedprior to the treating. In a further embodiment, the cytokine is IL-10 orTGF-β. In yet another embodiment, the treating results in an increasednumber of T-regulatory cells in the subject, as compared to the numberof T-regulatory cells prior to the treating.

In one embodiment of the method of treating food allergy providedherein, the treating results in an increase in the eliciting dose of theallergen, as compared to the eliciting dose prior to initiation oftreatment. For example, in one embodiment, the increase in the elicitingdose of the allergen is an increase by 10%, by 20%, by 30%, by 40%, by50%, by 60%, by 70%, by 80%, by 90%, by 100%, by 500%, by 1000%, by atleast about 10%, by at least about 20%, by at least about 30%, by atleast about 40%, by at least about 50%, by at least about 60%, by atleast about 70%, by at least about 80%, by at least about 90%, by atleast about 100%, by at least about 500% or by at least about 1000%.

In one embodiment of the method of treating food allergy providedherein, the treating results in a sustained unresponsiveness to theallergen. In a further embodiment, the sustained unresponsiveness lastsfor about 1 month, about 2 months, about 3 months, about 4 months, about5 months, about 6 months, about 7 months, about 8 months, about 9months, about 10 months, about 11 months, about 12 months, at leastabout 1 month, at least about 2 months, at least about 3 months, atleast about 4 months, at least about 5 months, at least about 6 months,at least about 7 months, at least about 8 months, at least about 9months, at least about 10 months, at least about 11 months or at leastabout 12 months after therapy has ended.

In yet another aspect of the invention, a device is provided for thetreatment of a food allergy. The device comprises a microneedle arraycomprising a plurality of solid microneedles extending from a commonsubstrate. Each microneedle of the plurality has a base, shaft and tip,and at least one microneedle of the array is coated with a foodallergen. In a further embodiment, the substrate is adhesive on at leastone side. The substrate in one embodiment, is rigid. However, in anotherembodiment, the substrate is flexible.

In one embodiment of the device, the average length of the microneedlesin the array is from about 100 μm to about 1000 μm, as measured from thebase of the tip of the microneedles. In a further embodiment, theaverage length of the microneedles in the array is from about 100 μm toabout 900 μm, or from about 100 μm to about 800 μm, or from about 100 μmto about 700 μm, or from about 100 μm to about 600 μm, or from about 100μm to about 500 μm.

The device in one embodiment, comprises a microneedle array comprisingfrom about 20 to about 150 microneedles, for example, from about 20 toabout 150 microneedles, from about 30 to about 100 microneedles, or fromabout 40 to about 100 microneedles.

The device in one embodiment, comprises solid microneedles coated with agroundnut, peanut, milk, egg, tree nut, seed, fish, shellfish,crustacean, cereal, legume allergen, or a combination thereof.

The device in one embodiment, comprises solid microneedles coated with apeanut allergen. In a further embodiment, the peanut allergen cancomprise Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8,Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, a peptide fragment thereof,or a combination thereof. In a further embodiment, the peanut allergencomprises Ara h1, Ara h2, Ara h6, a peptide fragment thereof, or acombination thereof. Additionally, where a peanut allergen is coated onthe microneedle array, it can be provided as peanut protein extract orpeanut flour or a combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 illustrates one embodiment of a process for assembling amicroneedle patch including coated in-plane microneedle rows asdescribed herein.

FIG. 2 is a cross sectional view of microneedles in a microneedle arraywhich are dipped into an allergen coating liquid using a physical maskto control deposition of coating, with the mask having multiple dipholes built into the mask.

FIG. 3 is a cross sectional view of microneedles in a microneedle arraywhich are dipped into an allergen coating liquid using a physical maskto control deposition of coating, with a single reservoir in fluidcommunication with open dip holes.

FIG. 4 is a scanning electron micrograph of one embodiment of amicroneedle array.

FIG. 5 is a fluorescent micrograph of a microneedle array prior toinsertion into the skin. The insets are of a single microneedle prior toinsertion (left) and after insertion (right).

FIG. 6 is a bar graph showing the amount of ovalbumin (Ova) deliveredinto mouse skin via microneedles, as well as the amount of Ova remainingon microneedles and on the skin surface. Ova was quantified viafluorescein-conjugated Ova (λex437 nm/λem515 nm) throughspectrofluorometer.

FIG. 7 shows the schedule and doses for sensitization and oral peanutchallenge in Balb/c mice with peanut extract (PE).

FIG. 8 (left) is a graph showing the body temperature (° C.) as afunction of time for naïve and sensitized mouse groups, after oralchallenge with PE, as measured with a rectal probe. FIG. 8 (right) is agraph of the anaphylactic score as a function of time in naïve andsensitized mouse groups after oral challenge with PE. An anaphylacticscoring system based on mouse activity was used to evaluate anaphylacticseverity as described previously by Li and McCaskill (Li et al. (1999).J Allergy Clin Immunol 103(2 Pt 1), pp. 206-14; McCaskill et al. (1984).Immunology 51(4), pp. 669-77, the disclosure of each of which is herebyincorporated by reference in its entirety for all purposes). 0: Nosymptoms; 1: Hypersensitivity to touch, irritation/aggression; 2:Puffiness around the eyes, pilar erection, reduced activity withincrease respiratory rate; 3: Cyanosis around the mouth and tail,labored breathing, lying flat; 4: Loss of consciousness, no activityupon prodding, tremor or convulsions; 5: Death. All data illustrated asmean±SEM. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

FIG. 9 (left) is a graph of PE-specific IgE antibodies in naïve andsensitized mouse groups after oral challenge with PE. FIG. 9 (right) isa graph showing histamine release (concentration in plasma pg/mL) innaïve and sensitized mouse groups after oral challenge with PE. All dataillustrated as mean±SEM. *p<0.05, **p<0.01.

FIG. 10 is an immunotherapy schedule employed with peanut coatedmicroneedles. Schedule shows the effect of PE dose on immune responseafter vaccination with PE coated MNs. Microneedles coated with 1, 5 or25 μg PE were used at one dose per week. Six weeks later (at day 56),mice were bled to check for anti-PE responses.

FIG. 11 (top left) is a graph of anti-PE IgG antibodies, as measured byELISA at different serum dilutions. FIG. 11 (top right) is a graph ofanti-PE IgG1 antibodies, as measured by ELISA at different serumdilutions. FIG. 11 (bottom left) is a graph of anti-PE IgG2a antibodies,as measured by ELISA at different serum dilutions. FIG. 11 (bottomright) is a graph of anti-PE IgE antibodies, as measured by ELISA atdifferent serum dilutions.

FIG. 12 is a bar graph showing PE specific antibody response for thethree microneedle groups. At day 56, mice were euthanized and bonemarrow was aseptically collected. Bone marrow cells were cultured inRPMI medium containing penicillin-streptomycin and 10% FBS. After 72hr., supernatant was collected to analyze PE specific antibodies usingELISA. Data illustrated as mean±SEM; ns: not significant.

FIG. 13 are graphs showing cytokine response of mice after splenocyterestimulation (IL-2, top left; IFN-7, top right; IL-4, bottom left;IL-5, bottom right). At day 56, mice were euthanized and spleen wasaseptically collected. Splenocytes were restimulated with PE (200 μg/ml)in an in vitro culture. After 12 hr. of restimulation, supernatant wascollected to analyze IL-2, while other cytokines were analyzed after 72hr. of restimulation. All data are illustrated as mean±SEM. *p<0.05, andns: not significant.

FIG. 14 is a microneedle immunotherapy schedule. Mice were sensitizedorally to peanut and treated with 5 μg PE coated on microneedles(MNs)-CIT (cutaneous immunotherapy) every week. Three weekspost-immunotherapy, mice were challenged orally with high dose of PE (20mg: 10 mg+10 mg at 30 min interval).

FIG. 15 are graphs of anti PE antibodies in plasma (at differentdilutions) after oral challenge. IgG—left graph. IgG1—right graph.Plasma was collected 5 minutes post challenge.

FIG. 16 are graphs of anti PE antibodies in plasma (at differentdilutions) after oral challenge. IgG2a—left graph. IgE—right graph.Plasma was collected 5 minutes post challenge.

FIG. 17 are graphs showing various anaphylactic indicators for mousetreatment groups (after oral challenge with peanut extract (PE)). FIG.17, top left: Post PE oral challenge, mice were monitored and theirsymptoms were scored and are shown as ‘anaphylactic score’; 0: nosymptoms; 1: hypersensitivity to touch, irritation/aggression; 2:puffiness around the eyes, pilar erection, reduced activity withincrease respiratory rate; 3: cyanosis around the mouth and tail,labored breathing, lying flat; 4: loss of consciousness, no activityupon prodding, tremor or convulsions; 5: death. FIG. 17, top right:Anaphylaxis mediator histamine in plasma collected 5 minutes afterchallenge for the four mouse groups. FIG. 17, bottom left: Anaphylaxismediator mast cell protease 1 (MCPT-1) in plasma collected 5 minutesafter challenge for the four mouse groups. All data are illustrated asmean±SEM. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

FIG. 18 are graphs for anti-ovalbumin (Ova) response in mice treatedwith Ova coated MNs with or without stimulator of interferon genes(STING) ligand adjuvants. Mice were treated at day (d) 0 and d28 withOva (25 μg) with or without STING adjuvants cGMP (25 μg) or cAMP (25 μg)using coated MNs. Serum was collected on d28 and d60 to determineanti-Ova antibody response using the ELISA method. (FIG. 18, Left)anti-Ova IgG and (FIG. 18, Right) anti-Ova gG2a response in mice serum.Individual mouse serum was diluted to 1:100 and used in analysis. Alldata illustrated as mean±SEM. ***p<0.0005, **** p<0.0001 and ns; notsignificant. ELISA: Enzyme-linked immunosorbent assay.

FIG. 19 are graphs for anti-Ova response in mice treated with Ova coatedMNs with or without CpG adjuvant. Mice were treated on day (d) 0, d7,d14 with Ova (25 μg)±CpG (25 μg) and bled at d21 and d35 to determineanti-Ova antibody response using the ELISA method. (FIG. 19, Left)anti-Ova IgG, and (FIG. 19, Right) anti-Ova IgG2a response in miceserum. Individual mouse serum was diluted at 1:20 dilution and used inanalysis. All data illustrated as mean±SEM. ***p<0.0005, **** p<0.0001and ns; not significant. ELISA: Enzyme-linked immunosorbent assay.

FIG. 20 are graphs for anti-peanut extract (PE) response in mice treatedwith PE coated MNs with or without CpG adjuvant. Mice were treated onday (d) 0, d7, and d14 with PE (25 μg)±CpG adjuvant (25 μg) coated onMNs. Serum was collected on d56 to determine anti-Ova antibody responsewith ELISA method. (FIG. 20, Left) anti-PE IgG, (FIG. 20, Middle)anti-PE IgG2a and (FIG. 20, Right) anti-PE IgE response in mouse serum.Individual mouse serum was used in analysis. All data illustrated asmean±SEM. .*p<0.05, **p<0.005 and ns; not significant.

FIG. 21 shows a peanut allergy immunotherapeutic schedule for examiningeffect of adjuvant. Immunotherapy schedule; mice were sensitized orallyevery week up to six weeks with 1 mg peanut extract (PE)+10 μg choleratoxin (CT). Three weeks later, sensitized mice were treated with MNscoated with PE (5 μg)±CpG (5 μg). Four weeks post-immunotherapy, micewere challenged orally with a high dose of PE (20 mg).

FIG. 22 shows therapeutic efficacy of CpG adjuvant in peanut allergytreatment. Five minutes after oral peanut challenge, mice were assessedfor allergic reaction. FIG. 22, Left: ‘Anaphylactic score’; 0: nosymptoms; 1: hypersensitivity to touch, irritation/aggression; 2:puffiness around the eyes, pilar erection, reduced activity withincrease respiratory rate; 3: cyanosis around the mouth and tail,labored breathing, lying flat; 4: loss of consciousness, no activityupon prodding, tremor or convulsions; 5: death. FIG. 22, Middle:Histamine level in serum. FIG. 22, Right: MCPT-1 level in serum. Alldata illustrated as mean±SEM. ****p<0.0001 and ns; not significant.

FIG. 23 (left) is a stereomicroscope brightfield image of a microneedlearray whose alternate diagonal rows are coated with two different dyes(green fluorescent fluorescein isothiocyanate and red fluorescentsulforhodamine) to simulate an allergen and an adjuvant coating onseparate rows of microneedles: FIG. 23 (right) is a stereomicroscopefluorescent and brightfield mixed-light image of the same array. Scalebar for both images is 500 μm

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

Peanut allergy is a life-threatening condition. About 1% of the USpopulation (˜3 million people) has peanut allergies, and there is noFDA-approved treatment. As such, strict avoidance, i.e., a peanut-freediet, is the only option available to manage peanut allergies. Moreover,although oral immunotherapy, classical allergy shots and transdermalpatches have all been attempted for the treatment of peanut allergy,each has drawbacks such as the lack of sustained unresponsiveness, andthe presence of adverse events.

Specifically, a major limitation of oral immunotherapy is that thepeanut oral dose is escalated to thousands of milligrams, which cancause various side effects. Additionally, peanut oral immunotherapy hasonly been shown in some instances to offer short term desensitization.Moreover, allergy shots, when administered for peanut allergy, resultedin systemic reactions after rush immunotherapy. Transdermal patches,currently being developed, are also met with challenges. Appreciatingthat skin is impermeable to large molecules such as proteins (see, e.g.,Karande and Mitragotri (2010). Annu. Rev. Chem. Biomol. Eng. 1, pp.175-201; Prausnitz et al. (2004). Nat. Rev. Drug Discov. 3(2), pp.115-124, the disclosure of each of which is incorporated by reference inits entirety for all purposes), the delivery of peanut proteins fromsuch a skin patch is in all likelihood very low, and as such, it is notsurprising that the immune modulating effect is not that strong. Indeed,none of the patients is the transdermal patch study could successfullycomplete the oral food challenge of 1044 mg (Jones et al. (2017). JAllergy Clin Immunol. 139(4), pp. 1242-1252, the disclosure of which isincorporated by reference in its entirety for all purposes).

The present invention addresses the need in the art for a new treatmentmethod for food allergy, and specifically, peanut allergy by providingmicroneedle arrays for the application to a patient's skin. Microneedles(MNs) are sharp microstructures, and due to their small size, MNs can benon-invasive and painless. Due to their micrometer dimensions, coatedMNs also have the potential to allow targeting of the allergens todendritic cells, e.g., Langerhans cells (LCs) that reside in the topmosthundred micrometers of the skin epidermis.

Skin dendritic cells (DCs) play a central role in the initiation ofallergic skin responses. Following encounter with an allergen, DCsbecome activated and undergo maturation and differentiate intoimmunostimulatory DCs and are able to present antigens effectively to Tcells. (Toebak et al. (2009). Contact Dermatitis 60(1), pp. 2-20,incorporated by reference herein in its entirety for all purposes).Without wishing to be bound by theory, it is thought that because MNscan target dendritic cells, e.g., LCs in the skin, they can help in dosereduction of the respective food antigen, e.g., peanut antigen.

In one aspect, the present invention is directed to a method fortreating a food allergy in a subject in need thereof. In one embodiment,the method for treating the food allergy comprises delivering aneffective amount of an allergen associated with the food allergy intothe subject's cutis. The delivering step comprises inserting a one ormore microneedles (e.g., present as a microneedle array) into thesubject's cutis, wherein the one or more microneedles each has abase,shaft and tip. At least one microneedle of the one or more microneedlesis coated with allergen associated with the food allergy and the atleast one coated microneedle of the array does not extend beyond thecutis once inserted into the subject's skin. However, in someembodiments described herein, each microneedle of the array does notextend beyond the cutis once inserted. In a further embodiment,substantially all the microneedles are coated with the allergen. Theallergen is allowed to dissociate from the one or more microneedleswhile inserted in the subject's cutis. Once the allergen dissociates,the one or more microneedles is removed from the subject's skin.

In one embodiment, the microneedle tips of the one or more microneedlesextend into the epidermis layer of the subject's skin once inserted. Ina further embodiment, the microneedle tips extend into the epidermislayer of the skin and do not extend into the dermis layer once insertedinto the subject. However, in some embodiments, the microneedle tips doextend into the dermis layer. It should be noted that unlike asubcutaneous injection, the microneedles provided herein do not extendbeyond the dermis layer of the skin, i.e., the microneedles do notextend into the subcutis. Additionally, microneedles of the one or moremicroneedles can be fabricated having different lengths. As a result,different microneedles can extend to different depths in the cutis.

The one or more microneedles can be present as an array of two or moremicroneedles, i.e., as a microneedle array. The one or more microneedles(e.g., microneedle array) comprises at least one solid microneedlecoated with or associated with one or more food allergens that arespecific to the food allergy. In one embodiment, substantially all ofthe microneedles in the array are coated with the one or more foodallergens. In another embodiment, a majority of the microneedles in thearray are coated with the one or more food allergens.

Because microneedles are very small structures, they are painless. Inembodiments of the treatment methods provided herein, a microneedlearray is inserted into the subject's skin one or more times for a timesufficient to cause a protective immune response, e.g., desensitizationor sustained unresponsiveness to the allergen. The allergen dose can bethe same or different for each insertion/application. For example,microneedle arrays can be applied serially, and deliver an escalatingdose of the allergen, or combinations of allergens.

The one or more microneedles (e.g., microneedle array) in one embodimentis inserted into a subject's skin one or more times for about 1 min. to1 hr., for example, from about 1 min. to about 10 min., or from about 1min. to about 5 min for each insertion (also referred to as anapplication). Where the one or more microneedles is inserted into asubject's skin multiple times (i.e., multiple applications), in oneembodiment, there is an “off period” in between the multipleapplications/insertions. The “off period” in one embodiment, is 12 hrs.,one day, two days, three days, four days, five days, six days, sevendays or 14 days. As such, the one or more microneedles (e.g., themicroneedle array) can be applied at various frequencies untildesensitization and/or sustained unresponsiveness to the allergen isachieved. In one embodiment, the one or more microneedles (e.g.,microneedle array) is inserted into a subject's skin once daily, twicedaily, every other day, every third day, or once a week until aprotective immune response is achieved. The time sufficient to achievedesensitization and/or long term unresponsiveness is 1 month, 3 months,6 months, 12 months, 18 months, 24 months 30 months or 36 months.

As used herein, the term “subject” is used to mean an animal, forexample a mammal, including a human or non-human. The terms subject andpatient can be used interchangeably. The subject can be a child or anadult. In one embodiment, a subject is from about 2 to about 30 yearsold. In a further embodiment, the subject is human. In anotherembodiment, the subject is human and is from about 2 years old to about12 years old. In a further embodiment, the subject is a human subjectand is from about 4 years old to about 11 years old or about 4 years oldto about 10 years old.

As used herein, the term “treating” or “treatment” refers to the abilityto achieve desensitization to the respective allergen, and/or long termunresponsiveness (also referred to as sustained unresponsiveness). Inone embodiment, the desensitization is characterized relative to thesame subject, prior to commencing therapy, or compared to a subjectreceiving placebo or not receiving treatment. In one embodiment, thesubject is desensitized by at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, about 70%, about 75%, at least about 80%, atleast about 85%, or at least about 90% as compared to the subject priorto commencing therapy, a subject receiving a placebo or a subject notreceiving treatment.

An “effective amount” of an allergen is an amount of allergen that canprovide desensitization to the allergen, and/or the increase ineliciting dose of the allergen. The effective amount can be delivered ina single treatment step, or as part of a treatment regimen wheremultiple doses are given during the treatment regimen. The treatmentregimen can include substantially the same dose for each allergenadministration, or can comprise at least one, at least two or at leastthree escalating dosages.

Successful desensitization can be characterized in one embodiment, by adecrease in the number of allergen specific IgE antibodies, and/orincreased production of T regulatory cells. The T-regulatory cells inone embodiment, are Tr1 cells (produce IL-10, IL-10+), (ii) Th3 cells(produce TGF-β, latency associated peptide:LAP+), (iii)CD4+CD25+forkhead box P3:Foxp3+ Tregs, or a combination thereof.

In another embodiment, successful desensitization is characterized by anincrease in cytokine production (e.g., IL-10, TGF-β), increasedproduction of IgG allergen specific antibodies (e.g., IgG4 in humans,IgG2a in mice), decreased number of mast cells (e.g., at the site ofallergen exposure (e.g., the gastrointestinal tract (GI) in the case offood allergens) as compared to prior to treatment), decreased number ofbasophils (e.g., at the site of allergen exposure (e.g., thegastrointestinal tract (GI) in the case of food allergens), or acombination of the foregoing.

Successful treatment can also be measured by an increase in theeliciting dose of the allergen, as compared to the eliciting dose priorto initiation of treatment. The “eliciting dose” of an allergen orallergenic food, as used herein, is the lowest dose of allergen orallergenic food containing the allergen, that causes a response in asubject that is sensitized to the allergen, e.g., symptoms of anallergic reaction. “Eliciting dose” can also be used interchangeablywith “threshold dose”. The symptoms can be skin inflammation/redness,upper airway (eyes, nose, and throat), lower airway (lungs),gastrointestinal, cardiovascular and/or neurological symptoms, asassessed by one of ordinary skill in the art. In one embodiment, thesymptom is a mild, objective symptom in a sensitized subject, e.g., ahighly sensitized subject. See, e.g., Taylor et al. (2004). Clin Exp.Allergy 34, pp. 689-695, the disclosure of which is incorporated hereinin its entirety for all purposes.

Low dose challenges can begin, e.g., at 10 μg of the allergen and cancontinue to increase based on the judgement of one of ordinary skill inthe art. In one embodiment, a 30 minute or 1 hr. interval is usedbetween doses. In one embodiment, the dose increase is an increase in anorder of magnitude.

In one embodiment, a peanut allergen challenge comprises theadministration of a peanut flour to a subject. The peanut flour can bedefatted, and can comprise Florunner, Virginia, or Spanish peanut flour,or a combination thereof. In one embodiment, the peanut flour comprisesequal parts Florunner, Virginia and Spanish peanut flour. In anotherembodiment, roasted peanuts are used as the challenge material. Theforegoing compositions can also be used to coat the microneedlesprovided herein.

“Long term unresponsiveness” and “sustained unresponsiveness” are usedinterchangeably herein, and refers to the lack of clinical reactivity tothe ingested food allergen for 1 month to 1 year after therapy hasended. In one embodiment, the sustained unresponsiveness lasts for about1 month, about 2 months, about 3 months, about 4 months, about 5 months,about 6 months, about 7 months, about 8 months, about 9 months, about 10months, about 11 months or about 12 months after therapy has ended. Inone embodiment, the sustained unresponsiveness lasts for at least about1 month, at least about 2 months, at least about 3 months, at leastabout 4 months, at least about 5 months, at least about 6 months, atleast about 7 months, at least about 8 months, at least about 9 months,at least about 10 months, at least about 11 months or at least about 12months after therapy has ended.

In one embodiment, the food allergy is a milk, fish, shellfish or nutallergy. In one embodiment, the food allergy is a nut allergy. In afurther embodiment, the nut allergy is a soy or a peanut allergy. Ineven a further embodiment, the nut allergy is a food allergy.

The one or microneedles (e.g., present as a microneedle array) providedherein can be used to delivery one or more food allergens to a subjectin need thereof in order to desensitize the subject to the allergen,and/or to obtain a sustained unresponsiveness to the allergen.

The term “allergen” refers to an immunogenic molecule (or a combinationthereof) involved in an allergic reaction contained in food. Theallergen in one embodiment, is a lipid, carbohydrate, protein, peptide,polypeptide, or a combination thereof. In one embodiment, the allergenis a native food preparation, a food extract, or a purified protein,polypeptide and/or peptide composition. The allergen may be in a naturalstate, or produced artificially (e.g., by recombinant and/or enzymatictechniques, and or de novo synthesis for instance). The allergen in oneembodiment, is structurally altered or modified to improve its stabilityor immunogenicity. The allergen in on embodiment is in admixture withone or more other constituents, such as an adjuvant. The allergen may bea mixture of several molecules (e.g., an extract such as a peanutprotein extract). The allergen may be present in combination with otherallergens, or in combination with other molecules from the food that arenot immunogenic. In one embodiment, one or more adjuvants are includedin a composition comprising the allergen.

The invention may be used with any food or food allergens such as,without limitation, groundnut, peanut, milk, egg, tree nuts and seeds(such as but not limited to: hazelnut, cashew, walnut, pecan, brazilnut, macadamia, chestnut, pistachio, coconut, almond, sesame, mustard),fish, shellfish, crustaceans, cereals (e.g., wheat, corn, oat, barley,rye, rice, sorghum, spelt), legumes (e.g., soy, kidney bean, black bean,common bean, chickpea, pea, cow pea, lentils, lupine), or mixturesthereof.

In one embodiment, the allergen is a peanut allergen or a combination ofpeanut allergens. The peanut allergen in one embodiment is in the formof a peanut protein extract. Thirteen peanut allergens (Ara h1 throughAra h13) have been recognized by the Allergen Nomenclature Sub-Committeeof the International Union of Immunological Societies (Zhou et al.(2013). International Journal of Food Science, V. 2013, Article ID909140, incorporated by reference herein in its entirety). In oneembodiment, the peanut allergen comprises one or more of Ara h1, Ara h2,Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Arah11, Ara h12 or Ara h13, or a peptide fragment of one of the foregoing,or a combination thereof. In a further embodiment, the peanut allergencomprises Ara h1, Ara h2, Ara h3, a peptide fragment thereof, or acombination thereof. In yet another embodiment, the peanut allergencomprises Ara h1, a peptide fragment thereof, or multiple peptidefragments thereof.

Peanut Flour (PF) for use as an allergen composition can be obtainedcommercially, for example, from the Golden Peanut Company (Alpharetta,Ga.). The PF can be defatted, and can comprise in one embodiment,Florunner, Virginia, or Spanish PF, or a combination thereof. In oneembodiment, the peanut flour comprises equal parts Florunner, Virginiaand Spanish PF. In another embodiment, roasted peanuts are used as asource of allergen for the allergen composition. Peanut extract for useas an allergen composition in another embodiment, can be obtainedcommercially, for example, from Greer Labs (Lenoir, N.C.).

In one embodiment, the peanut allergen comprises Ara h1 (or a peptidefragment thereof), Ara h2 (or a peptide fragment thereof), and Ara h6(or a peptide fragment thereof).

Representative linear epitopes for peanut allergens are provided in Zhouet al. (Zhou et al. (2013). International Journal of Food Science, V.2013, Article ID 909140, incorporated by reference herein in itsentirety). For example, for Ara h1, epitope sequences that can beincorporated into the peanut allergen include PGQFEDFF (epitope #7),YLQGFSRN (epitope #8), FNAEFNEIRR (epitope #9), QEERGQRR (epitope #10),DITNPINLRE (epitope #11), NNFGKLFEVK (epitope #12), GNLELV (epitope#13), RRYTARLKEG (epitope #14), ELHLLGFGIN (epitope #15), HRIFLAGDKD(epitope #16), IDQIEKQAKD (epitope #17), KDLAFPGSGE (epitope #18),KESHFVSARP (epitope #19), NEGVIVKVSKEHVEELTKHAKSVSK (epitope #21), or acombination thereof.

Peptides that may be incorporated into an Ara h2 peanut allergen includeHASARQQWEL (epitope #1), QWELQGDRRC (epitope #2), DRRCQSQLER (epitope#3), LRPCEQHLMQ (epitope #4), KIQR.DEDSYE (epitope #5), YERDPYSPSQ(epitope #6), SQDPYSPSPY (epitope #7), DRLQ..GRQQEQ (epitope #8),KRELRNLPQQ (epitope #9), QRCDLDVESG (epitope #10), or a combinationthereof.

Peptides that may be incorporated into an Ara h3 allergen includeIETWNPNNQEFECAG (epitope #1), GNIFSGFTPEFLAQA (epitope #2),VTVRGGLRILSPDRK (epitope #3), DEDEYEYDE-EDRRRG (epitope #4), or acombination thereof.

In one embodiment, the allergen is a legume allergen or a tree nutallergen. For example, the allergen in one embodiment is soy. In anotherembodiment, the allergen is almond, pecan, hazelnut, walnut or acombination thereof. It should be noted that certain patients aresensitized against more than one type of food allergen (Sicherer et al.(1998). Pediatrics 102(1), p. e6; Sicherer et al. (2001). J Allergy ClinImmunol. 108(1), pp. 128-132, each of which is incorporated by referenceherein in its entirety for all purposes). As such, some embodiments ofthe invention are directed to the delivery of multiple allergens to apatient in the treatment methods provided herein. Alternatively, anallergen is cross reactive to two different food substances, andtherefore, in one embodiment, a cross reactive allergen can be used todesensitize a patient to multiple food allergens. In cross-reactivity,IgE antibodies against one allergen can bind to a different homologousallergen and trigger the adverse reaction similar to that elicited byits binding to the first allergen. Homologous allergens share structuralsimilarity or common epitopes, which increases the chances ofcross-reactivity. For example, peanut proteins share structural homologywithin the legume family (e.g. soy protein), and with certain tree nuts(e.g. almond, pecan, hazelnut, and walnut) (Sicherer et al. (2000).Allergy 55(6), pp. 515-521; de Leon et al. (2003). Clin. Exp. Allergy33(9), pp. 1273-1280; Rosenfeld et al. (2012). Int. Arch. AllergyImmunol. 157(3), pp. 238-245, each of which is incorporated by referenceherein in its entirety for all purposes).

The one or more allergens provided herein are delivered to a subject inneed thereof in an allergen composition. The allergen compositionincludes at least one allergen in a pharmaceutically acceptable vehicle.In one embodiment, the allergen composition further comprises one ormore adjuvants.

An “adjuvant” is substance added to one of the allergens provided hereinto increase the allergen's immunogenicity, as compared with itsimmunogenicity in absence of the adjuvant. An adjuvant may be includedin an allergen composition provided herein, for example, to increase theefficacy of the allergen and/or to induce or enhance an immune responsethat is not sufficiently induced in the absence of the adjuvant. In someembodiments, the adjuvant enables a lower dose of the allergen. Inanother embodiment, the adjuvant enables a more rapid immune response.The practical result of the more rapid immune response is a reduction ina multi-dosing regimen to a fewer number of doses, and in some cases, asingle dose.

The adjuvant can be mixed with the allergen and present in the samemicroneedle coating. Alternatively, the adjuvant and allergen can becoated on separate microneedles. In one embodiment where an adjuvant isdelivered with an allergen with a microneedle array, the allergen andadjuvant are coated on different microneedles of a microneedle array. Ina further embodiment, individual rows of a microneedle array are coatedwith either the allergen or the adjuvant. See, e.g., FIG. 23 left andright.

FIG. 23 (left) is a stereomicroscope brightfield image of a microneedlearray whose alternate diagonal rows are coated with two different dyes(green fluorescent fluorescein isothiocyanate and red fluorescentsulforhodamine) to simulate an allergen and an adjuvant coating onseparate rows of microneedles: FIG. 23 (right) is a stereomicroscopefluorescent and brightfield mixed-light image of the same array. Scalebar for both images is 500 μm.

In one embodiment, the adjuvant is an aluminum salt, inulin, L-Tyrosine,algammulin, combination of inulin and aluminum hydroxide, monophosphoryllipid A (MPL), L-Tyrosine in combination with MPL, resiquimod, muramyldipeptide (MDP), N-glycolyl dipeptide (GMDP), poly IC, CpGoligonucleotide, resiquimod, aluminum hydroxide with MPL, any water inoil emulsion, any oil in water emulsion that contains one or more of thefollowing constituents: squalene or its analogues or anypharmaceutically acceptable oil, tween-80, sorbitan trioleate,alpha-tocopherol, cholecalciferol, calcium phosphate or a combination oftwo or more of the foregoing.

In one embodiment of the allergen composition provided herein, thecomposition comprises a stimulator of interferon genes (STING) ligandadjuvant. The STING ligand in one embodiment, is a cyclic dinucleotideor a xanthenone derivative. In a further embodiment, the STING ligand iscyclic guanosine monophosphate (cGMP), cyclic di-GMP (c-diGMP), cyclicadenosine monophosphate (cAMP), cyclic-di-AMP (c-di-AMP), cyclic-GMP-AMP(cGAMP, e.g., 2′2′-cGAMP, 2′3′-cGAMP or 3′3′-cGAMP). STING ligands areavailable commercially, e.g., from Invivogen (San Diego, Calif., USA).

In another embodiment, the adjuvant is an oil and water emulsion (forexample, complete Freund's adjuvant and incomplete Freund's adjuvant,Corynebacterium parvum, Bacillus Calmette Guerin, aluminum hydroxide,glucan, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant,certain synthetic polymers such as poly amino acids and co-polymers ofamino acids, saponin, Avridine (N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), paraffin oil,muramyl dipeptide or a combination thereof.

In one embodiment, the allergen composition comprises alum as anadjuvant.

In one embodiment, the adjuvant in the allergen composition isL-Tyrosine. Various animal studies have shown L-Tyrosine to be a safeand effective adjuvant, with high adsorptive power for proteins, andenhancement of antibody indication as well as a short-term depot. See,e.g., Baldrick et al. (2002). J. Appl. Toxicol. 22, pp. 333-344, thedisclosure of which is incorporated by reference herein in its entiretyfor all purposes.

In another embodiment, the adjuvant in the allergen composition ismonophoshoryl lipid A (MPL).

In another embodiment, the adjuvant in the allergen composition isL-Tyrosine in combination with monophoshoryl lipid A (MPL).

In one embodiment, the adjuvant in the allergen composition is a CpGoligonucleotide (ODN). For example, the CpG adjuvant is one or moreadjuvants disclosed in U.S. Patent Application Publication No.2017/0136119, the disclosure of which is incorporated by reference inits entirety for all purposes.

In yet another embodiment, the allergen is delivered with an adjuvantselected from alum; a CpG oligonucleotides (ODN); polyA-polyU;dimethyldioctadecylammonium bromide (DDA),N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)propanediamine, carbomer,chitosan (see, e.g., U.S. Pat. No. 5,980,912 for example, the disclosureof which is incorporated herein by reference in its entirety for allpurposes.

The adjuvant, in another embodiment, comprises a lipophile, a polymer ofacrylic or methacrylic acid, saline, cholesterol, a saponin, sodiumhydroxide, or a combination thereof. For example, one or more of theadjuvants disclosed in U.S. Patent Application Publication No.2017/0202959 and U.S. Pat. No. 9,730,987, the disclosure of each ofwhich is incorporated herein by reference in its entirety for allpurposes.

The devices and methods provided herein employ one or more microneedlesto deliver an allergen into a subject's cutis. In one embodiment, asingle microneedle is employed. However, in another embodiment, two ormore microneedles are employed. The two or more microneedles can be inthe form of a microneedle array. A microneedle patch in embodimentsdescribed herein, includes one or more microneedles extending from acommon substrate. Where two or more microneedles are employed, e.g., asan array, each microneedle need not be coated with allergen. However, inone embodiment, substantially every microneedle is coated with allergen.

“Microneedle array” as used herein, refers to two or more microneedlesextending from a common substrate. Each microneedle includes a base, atip portion and a shaft between the base and tip portion. The two ormore microneedles in the array need not be homogenous with respect tosize, shape and/or material. In other words, a microneedle array mayinclude a mixture of different microneedles. For example, an array mayinclude microneedles having various lengths, base portion diameters, tipportion shapes, spacings between microneedles, drug coatings, etc.However, in one embodiment, the two or more microneedles in the arrayare substantially the same size and shape, and are fabricated from thesame material. In a further embodiment, the two or more microneedles inan array are each fabricated from stainless steel, and are solidmicroneedles. In one embodiment, the microneedle comprises between 2 and1000 (e.g., between 2 and 500) microneedles. In one embodiment, themicroneedle array comprises between 2 and 250 microneedles, for example,between 2 and 100 microneedles, or from 10 to 100 microneedles.

The microneedles provided herein can be fabricated of differentbiocompatible materials, including metals, glasses, semi-conductormaterials, ceramics, or polymers. Examples of suitable metals includepharmaceutical grade stainless steel, gold, titanium, nickel, iron, tin,chromium, copper, alloys thereof, and combinations thereof. In oneembodiment, microneedles are fabricated from stainless steel.

In another embodiment, the microneedle is fabricated from a polymersubstrate. The polymer can be biodegradable or non-biodegradable.Examples of suitable biocompatible, biodegradable polymers includepolylactides, polyglycolides, polylactide-co-glycolides (PLGA),polyanhydrides, polyorthoesters, polyetheresters, polycaprolactones,polyesteramides, poly(butyric acid), poly(valeric acid), polyurethanesand copolymers and blends thereof. Representative non-biodegradablepolymers include polyacrylates, polymers of ethylene-vinyl acetates andother acyl substituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blends andcopolymers thereof. Biodegradable microneedles can provide an increasedlevel of safety compared to non-biodegradable ones, such that they areessentially harmless even if inadvertently broken off into thebiological tissue. In embodiments where biocompatible polymers areemployed, the allergen can either be coated on the surface of themicroneedle, or encapsulated with the polymer for example, as describedin PCT Publication WO 2014/182932, the disclosure of which isincorporated by reference herein in its entirety. For example, asolution of biocompatible polymer can be mixed with allergen and cast ina mold to form microneedles.

The microneedles employed herein can be solid or hollow. In addition,the microneedles can be porous or non-porous. The microneedles may beplanar, cylindrical, or conical. The microneedles can have a straight ortapered shaft. In one embodiment, the microneedle array comprises two ormore solid microneedles. In a further embodiment, each microneedle inthe array is a solid microneedle.

In one embodiment, the diameter of one or more of the microneedles inthe array is greatest at the base end of the microneedle (i.e., theportion attached to the substrate) and tapers to a point at the enddistal the base. In a further embodiment, each of the microneedles inthe array has a diameter that is greatest at the base, which tapers to apoint at the end distal to the base. The microneedles can also befabricated to have a shaft that includes both a straight (i.e.,untapered) portion and a tapered portion. In another embodiment, one ormore microneedles in the array are straight, while one or moremicroneedles are tapered. In yet another embodiment, the microneedles inthe array comprise shafts that have a circular cross-section in theperpendicular. However, in another embodiment, the microneedles in thearray comprise shafts that have a non-circular cross-section.

Each microneedle employed herein includes a tip portion. The tip portioncan have a variety of configurations. The tip portion can be symmetricalor asymmetrical about the longitudinal axis of the shaft. Moreover, thetip portion in one embodiment, is beveled, tapered, squared-off, orrounded. The tip portion, in one embodiment, has a length that is lessthan 50% of the total length of the microneedle.

Microneedle length selection, as an initial matter, is selectedconsidering whether the entire length of the microneedles is inserted,or whether a portion of the microneedles is inserted with a portion thatremains uninserted. The length of a microneedle is measured from thebase, i.e., the portion of the microneedle attached to the substrate, tothe tip of the microneedle. In the case of a microneedle array, wheretwo or more microneedles are employed, the microneedles can havesubstantially the same length, or different lengths. Different lengthscan be employed, for example, to deliver allergen to different depths inthe subject's cutis. In one embodiment, the average length of themicroneedles in a microneedle array is from about 50 μm to about 5000μm, from about 100 μm to about 1500 μm, from about 200 μm to about 1000μm, from about 200 μm to about 800 μm, from about 200 μm to about 700μm. In one embodiment, the average length of the microneedles in thearray is from about 500 μm to about 1000 μm. In yet another embodiment,the average length of the microneedles in the array is about 150 μm,about 250 μm, about 300 μm, about 500 μm, about 600 μm, about 700 μm,about 750 μm, about 800 μm or about 850 μm.

The cross-section of the microneedle, or width, is tailored to provide,among other things, the mechanical strength to remain intact for thedelivery of the drug or for serving as a conduit (i.e., in the case of ahollow microneedle), while being inserted into the skin, while remainingin place during its functional period, and while being removed (unlessdesigned to break off, dissolve, or otherwise not be removed). Invarious embodiments, the base portions of the microneedles in the arrayhave an average width or cross-sectional dimension from about 20 μm toabout 500 μm, for example from about 50 μm to about 350 μm, or fromabout 100 μm to about 250 μm. In one embodiment, the width of themicroneedles in the array is substantially the same in the base and theshaft of the microneedles.

The one or more microneedles, in one embodiment, have an average aspectratio (width:length) of from about 1:1 and 1:10. The tip of themicroneedle can sharpen gradually as in the case of microneedles with aconical, pyramidal, or triangular cross-section. In another embodimentthe tip can be suddenly formed into a sharp point as in the case ofmicroneedles with cylindrical cross-section. In one embodiment, themicroneedles have an aspect ratio of about 1:3.5 with a cross-sectionthat is rectangular for about 70% of its length followed by a taperingtriangular shape constituting the remaining about 30% of the top, andculminating in to a sharp tip.

The one or more microneedles, in one embodiment, includes a microneedlecomprising a pocket. As used herein, “pocket” refers to an apertureextending crosswise into the microneedle shaft (e.g., perpendicular tothe direction of microneedle movement during the process of insertioninto skin). The pocket can extend through the shaft. However, the pocketin another embodiment, is closed at one end, distal the opening in theshaft. This is distinct from a hollow bore wherein a concentric spaceextends substantially through the axial length of the shaft. The pocketsare considered to be part of the surface of the microneedle. In oneembodiment, the pocket is included in a solid microneedle, and includescoating material which may be particularly advantageous in certainembodiments where the coating material needs to be protected frommechanical forces during the insertion process, e.g., when the coatingcomprises a liquid or particles. Without wishing to be bound by theory,it is thought that such coating materials are more likely than others tobe prematurely dislodged from the microneedle during insertion intoskin, diminishing the complete delivery of the complete dosage of thecoating. However, the pockets of the microneedles advantageouslyfunction to shield the coating material therein from the mechanicalforces of insertion. The pockets may be made in various shapes (e.g.,circular, square, rectangular) and of various numbers and dimensions anddifferent spacings within the microneedle.

The microneedles in the arrays provided herein can be fabricated by avariety of methods known in the art. In one embodiment, a wet etchprocess is employed. For example, the wet etch processes described in Maet al. (2014). Pharmaceutical Research 31(9), pp. 2393-2403; Jain et al.(2016). Journal of Controlled Release 239, pp. 72-81, each of which isincorporated by reference herein in its entirety for all purposes, canbe employed.

Details of other contemplated manufacturing techniques are described,for example, in U.S. Patent Application Publication No. 2006/0086689,U.S. Patent Application Publication No. 2006/0084942, U.S. PatentApplication Publication No. 2005/0209565, U.S. Patent ApplicationPublication No. 2002/0082543, U.S. Pat. Nos. 6,334,856, 6,611,707,6,743,211, each of which is incorporated herein by reference in itsentirety for all purposes.

In one embodiment, the microneedles are cut from stainless steel orother metal sheets using a laser (e.g., an infrared laser) or othertechniques known in the art.

In one embodiment, an electropolishing technique is used to produceclean, smooth, and sharp solid microneedles. Electropolishing can removeslag deposits from the microneedles, as laser-cutting of metals such asstainless steel may produce microneedles with rough edges covered withslag deposits. In one embodiment, laser cut stainless steel microneedlesare electropolished in a solution that includes glycerin,ortho-phosphoric acid (85%), and water. In one example, a copper plateis used as the cathode and the metal microneedles serve as the anode.The anode may be vibrated using means known in the art to help removegas bubbles generated at the anodic surface during electropolishing.Electropolishing is believed to be especially effective, because currentdensity (i.e., etching rate) is largest at sites of high curvature,which inherently targets sites of surface roughness for removal. In someembodiments, the electropolishing process has an output rate of finishedmicroneedle arrays of one 50-needle array every 30 minutes using asingle laser. This rate can be increased by process optimization and useof multiple lasers.

In one embodiment, the microneedle array used in the methods providedherein (or the single microneedle) includes a substantially planarfoundation from which two or more microneedles extend (or the singlemicroneedle extends), typically in a direction normal (i.e.,perpendicular or out-of-plane) to the foundation. Alternatively,microneedles may be fabricated on the edge of a substrate ‘in-plane’with the substrate. In one embodiment, the microneedle array extendsfrom a flexible base substrate. In another embodiment, the microneedlearray extends from a curved base substrate. The curvature of the basesubstrate typically would be designed to conform to the shape of thetissue surface. The curved base substrate can be flexible or rigid.

In one embodiment, the one or more microneedles extends from an adhesivepatch substrate. The patch comprises one or more microneedles, forexample, an array of tens or hundreds of microneedles (e.g., from about10 to about 500 microneedles or from about 10 to about 100microneedles). The patch, in one embodiment, comprises an adhesivecomponent to secure the patch to the skin. The patch includes aplurality of linear rows of in-plane microneedles, a plurality ofindividual arrays of out-of-plane microneedles, or a combinationthereof.

The patch, e.g., adhesive patch, can be a flexible or rigid substratewhich includes a pressure sensitive adhesive as known in the art.

In one embodiment, the microneedles and adhesive component areconfigured such that the microneedles extend through apertures in theadhesive layer. Individual microneedles or subgroups of microneedles(e.g., rows) can extend through a single aperture. Without wishing to bebound by theory, it is thought that when the adhesive surface isadjacent the microneedles, the adhesive is able to better hold themicroneedles down and to compensate for the recoiling-tendency of skinand/or a rigid substrate for out-of-plane microneedles.

In one embodiment, in-plane microneedles are fabricated with a uniformadhesive layer in between the microneedles. For example, rows ofmicroneedles can be assembled into a patch by forming slits (equal tothe length of an in-plane row) in a material, e.g., polyethylene medicalfoam tape. Such cutting can be performed by any suitable technique knownin the art, such as laser cutting. The microneedle rows can be manuallyor robotically inserted into each slit from the non-adhesive side of thefoam tape and glued to the foam tape using a medical grade adhesive. Theadhesive is then allowed to cure. Optionally, a medical foam tape ofsufficient thickness can then be cut into a disc and affixed onto thedried glue area to provide a cushioned backing to facilitate pressingthe patch during insertion. See FIG. 1. In one embodiment, the thicknessof the medical foam tape is from about 0.4 mm to about 1.0 mm, or fromabout 0.6 mm to about 1.0 mm, or from about 0.7 mm to about 0.9 mm,e.g., 0.8 mm.

In another embodiment, a microneedle patch is assembled using out-ofplane microneedles, a circular disc of a single-sided medical foam tapeand a thick double-sided medical tape. In the middle of the disc, arectangular piece of adhesive release liner equal in dimensions to theperiphery of the array can be cut out and peeled off. The microneedlearray can then be attached to this exposed adhesive. To provide a layerof pressure-sensitive adhesive on the stainless steel substrate of theaffixed array itself, a double-sided, carrier tape first perforated withholes corresponding to the microneedles can be attached by slipping itover the microneedles using an alignment device. In one embodiment, thecarrier tape is a polyethylene terephthalate (PET) carrier tape.

In one embodiment, microneedle array patches are assembled intotransdermal patches containing pressure-sensitive adhesive to adhere tothe skin. To secure microneedles in the skin at all times until ready tobe removed, microneedles in one embodiment, are integrated into aBand-Aid-like patch. The patch had pressure-sensitive adhesive on onecomplete side, with microneedles protruding therefrom. The adhesivesecured the microneedles and compensated for the recoiling tendency ofthe skin and the rigid stainless steel material of the out-of-planemicroneedles (i.e., microneedles normal to the patch substrate). Patchescan be fabricated using either multiple linear rows of in-planemicroneedles or individual arrays of out-of-plane microneedles.

In-plane microneedles, in one embodiment, are fabricated with a uniformadhesive layer in between the microneedles. In this embodiment, a set ofrows of microneedles (e.g., 10 rows), each containing, for example, 5-10microneedles each, can be assembled into a patch of, for example, 50-100microneedles. In one embodiment, slits are laser cut into a single sidedmedical foam tape. Each slit is cut to the length of a row ofmicroneedles, and the number of slits corresponds to the number ofmicroneedle rows in the patch. Microneedle rows can be manually orrobotically inserted into each slit from the non-adhesive side of thefoam tape, and glued to the foam tape using a medical grade adhesive.The adhesive can then be allowed to cure for a sufficient amount oftime, for example from about 12 hours to about 48 hours, for exampleabout 24 hours. A medical foam tape can then be cut to size of theassembled array, and affixed onto the dried glue area to provide acushioned backing to facilitate pressing the patch during insertion.

In one embodiment, a microneedle patch is assembled with out-of planemicroneedles. In this embodiment, a circular disc of appropriatediameter is cut from a single-sided medical foam tape, for example,using a CO₂ laser. One of ordinary skill in the art will appreciate thatthe diameter of the disk will be dictated by the size and shape (e.g.,number of rows) of the microneedle array. In the middle of this disc, arectangular piece of the adhesive release liner equal in dimensions tothe periphery of the array can be cut out, e.g., using a CO₂ laser, andsubsequently peeled off. The stainless steel microneedle array can thenbe attached to this exposed adhesive. To provide a layer ofpressure-sensitive adhesive on the stainless steel substrate of theaffixed array itself, a double-sided carrier tape (e.g., polyethyleneterephthalate (PET) tape) can be attached. The carrier film is firstperforated with holes at the same spacing as the microneedles using aCO₂ laser. The tape is then slipped over the microneedles using acustom-built alignment device and pressed to stick against the stainlesssteel microneedles.

The coated solid microneedles provided herein can be fabricated viamethods known to those of ordinary skilled in the art. For example, inone embodiment, the coated microneedles are fabricated via the methodsdisclosed in U.S. Pat. No. 9,364,426, the disclosure of which isincorporated by reference in its entirety for all purposes.

In one embodiment, prior to coating the microneedle or microneedles withallergen, the microneedle or microneedles are treated with oxygen or airplasma. Such treatment has been reported to increase the surface energyand wettability of certain substrates such as stainless steel. See,e.g., Tang et al. (2004). Korean J. Chem. Eng. 21(6), pp. 1218-1223, thedisclosure of which is incorporated by reference in its entirety for allpurposes. Moreover, an oxygen or air plasma treatment may result inadditives not being needed in the subsequent allergen coating (e.g.,additive to facilitate coating adhesion), and can serve to sterilize themicroneedle surface.

In some embodiment, prior to coating the one or more microneedles (e.g.,microneedle array) with the allergen or combination of allergens, aprecoating to at least one surface of the microneedles is performed, inorder to increase the surface energy of the surface, or to otherwisemodify the surface energy properties of the microneedles. In anotherembodiment, the coating liquid is modified to decrease the surfacetension of the coating liquid. A combination of the aforementioned canalso be carried out. It should also be noted that a precoating need notbe applied to all microneedles of the one or more microneedles. Nor doesthe coating liquid need to be modified for all microneedles, when amodification of the coating liquid is performed.

The coating liquid in one embodiment, is disposed in one or morereservoirs. Microneedles can be dipped directly into the reservoircontaining the allergen or combination of allergens. In anotherembodiment, a physical mask having a plurality of aperturestherethrough, each aperture having cross-sectional dimensions largerthan the at least one microneedle to be coated is provided over thereservoir. In this embodiment, the microneedle array is aligned with theplurality of apertures, and the array is inserted through the alignedaperture and into the coating liquid. The coated microneedle array isthen removed from the coating liquid and from the apertures. The one ormore reservoirs may be defined in a secondary structure or the physicalmay have a plurality of the reservoirs defined therein.

By utilization of a physical mask, access of the coating liquid isrestricted only to the microneedle shaft and tip, thereby preventingcontamination of the substrate from which the microneedles extend. Thus,any meniscus rise or capillary action that may cause contact of thecoating liquid to an adjacent microneedle or with the substrate isavoided such that the coating is on the surface of the microneedleshafts and tips, and the base substrate is free of the coating.

In one embodiment, the physical mask is in the form of a plate having aone or more discrete apertures therethrough. The apertures, in oneembodiment, are in the form of one or more holes or slits which closelycircumscribe each microneedle, a single row of microneedles, multiplerows of microneedles, or another subset of microneedles of the array. Asused herein, the term “closely circumscribe” means that the physicalmask is effective to restrain, for example, by surface tension forces,the coating liquid to the reservoir and apertures, preventing it from“climbing up” the microneedle shaft substantially beyond the dippedportion of the microneedle which it is desired to coat. Surface energyproperties of the coating system (physical mask, microneedle, andcoating fluid) and operating conditions (e.g., temperature,dipping/withdrawal speed) can impact the selection of appropriatedimensions for the holes and slits.

In one embodiment, the physical mask is in the form of a substantiallyrigid plate secured to the reservoir (see, e.g., FIG. 2). The plateincludes an array of micron-sized holes which are used for inserting themicroneedles to be coated. When aligned, for example usingmicropositioners or pre-aligned parts moving on a rail, each of themicroneedles can be simultaneously inserted through the micron sizedholes and into the coating liquid, resulting in a controlledmicro-dip-coating process. The use of one or more micropositioners canbe used to provide control over the microneedle length being coated,that is how much of the microneedle length is actually coated. In oneembodiment, physical stops in the form of thick sheets or protrudingcylinders in between the physical mask and microneedles, or acombination thereof, are used to control the microneedle length beingcoated. The coating device can be configured to coat singlemicroneedles, in-plane rows of microneedles, and out-of-plane arrays ofmicroneedles.

In another embodiment, the physical mask acts as a coating liquidreservoir or reservoirs. For example, in one embodiment, the physicalmask includes reservoirs, closed at one end, that can be filled with thecoating liquid (see, e.g., FIG. 3). Single microneedles or multiplemicroneedles of an array can be dipped into each reservoir. In oneembodiment, the apertures of the mask have a closed bottom, and thecoating liquid is filled in these apertures from the open top. However,an inlet port can be present on the bottom of the apertures to fillcoating liquid. Apertures can be periodically or continually refilled tomaintain a constant amount of coating liquid in the reservoir(s).

To reduce propensity of air bubbles in the reservoir and/or apertures inthe plate, vent holes designed to release entrapped air can be providedin the coating apparatus. To prevent evaporation of coating liquid (orsolvent thereof) from the coating liquid, a pumping device (e.g., anautomated or manually pulsated syringe plunger) can be included with thecoating apparatus to fill the coating liquid reservoir and tooscillate/mix the coating liquid in dip-coating holes. The coatingliquid in the reservoir may be flowed or agitated to facilitatemaintenance of a uniform coating liquid composition during the dippingprocess. Alternatively, or additionally, the coating process may beperformed at a reduced temperature (relative to ambient) to reduce therate of evaporation of the coating liquid or solvent portion thereof.

In one embodiment, the coating process includes the step of volatilizingat least a portion of the solvent to form a solid coating. This may bereferred to as “drying” the coating or coating liquid. A similar stepmay be included when using molten coating liquids, wherein the coatedliquid is permitted to (or actively caused to) cool the molten materialsufficiently to cause it to solidify, forming a solid coating on atleast a portion of the microneedles of the array.

Microneedles can be coated with a single coating or multiple coatings.For example, the coating method in one embodiment includes inserting atleast one coated microneedle of a coated microneedle array into the sameor a different coating liquid and then removing the microneedle fromsaid same or different coating liquid. The composition of the coatingliquid may include a solvent to dissolve part of the previous coating,if desired. In another embodiment, the coating method includes the stepof applying a second coating liquid onto the solid coating or onto asecond surface of the microneedle in need of coating. The composition ofthe second coating liquid may include a second antigenic epitope.Multiple such dippings into the same or a different coating liquid maybe repeated.

The coating process can also include an optional intervening dip into acleaning solvent, e.g., to thin or remove part of a coating layer. Thismay be useful to build complete coating structures, e.g., where onecoating composition is located on one part of the microneedle (e.g., afirst pocket) and a second coating composition is located on anotherpart of the microneedle (e.g., a second pocket).

To obtain uniform coatings on microneedle surfaces, it is generallydesired that the surface tension of the coating liquid is lower than thesurface energy of the microneedle surface material (material ofconstruction or overcoat deposition). A slow (taking more than a second)or rapid (taking less than a second, e.g., less than a tenth of a secondor less than a hundredth of a second) withdrawal of the microneedle fromthe immersed state to outside the coating liquid will provide a uniformcoating on the microneedle. Addition of a viscosity enhancer to thecoating solution increases the coating thickness by increasing the filmthickness of the entrained liquid during withdrawal. However, therequirement of coating liquid surface tension being lower than themicroneedle material can be overcome by conducting the coating processat a rate faster than is needed to achieve thermodynamic equilibrium.For instance, by increasing the viscosity and withdrawing at a rapidspeed, the microneedle will entrain a significant volume of the liquidon the surface. If the solvent then evaporates before the liquid filmcan contract to form an island in the middle of the microneedle surface,the solid coating will become uniformly deposited onto the microneedles.Another way to overcome the surface tension barrier to obtain uniformcoatings is to use a non-aqueous solvent that has lower surface tension,possibly lower than the microneedle material. Similarly, while coatingonly the pockets, advantage can be made of the kinetic effect byutilizing a high surface energy liquid/solution that will not wet themicroneedle surface but will fill the pockets. Again, the speed must besufficiently slow so that liquid does not entrain on the surface, butonly gets into the pockets.

EXAMPLES

The present invention is further illustrated by reference to thefollowing Examples. However, it should be noted that these Examples,like the embodiments described above, are illustrative and are not to beconstrued as restricting the scope of the invention in any way.

Example 1—Intradermal Delivery of Model Antigen Via Coated Microneedles

Microneedle arrays were fabricated from 50 μm-thick stainless steel(304) sheets using a wet etch process. Each microneedle measured about700 μm in length and about 200 μm in width, and each microneedle arraycontained 57 microneedles. Microneedle arrays were fabricated asdescribed previously (see, e.g., Ma et al. (2014). PharmaceuticalResearch 31(9), pp. 2393-2403; Jain et al. (2016). Journal of ControlledRelease 239, pp. 72-81, each of which is incorporated by referenceherein in its entirety for all purposes. The individual microneedleswere then manually bent to make them perpendicular to the metal sheet(FIG. 4). Microneedles were coated using a micro-precision dip coatingstation developed in-house. It comprised of an automated x-y linearcomputer-controlled stage on to which microneedles were mounted. Thecoating solution was housed in an orifice in to which the microneedleswere dipped through motion control of the x-y stage, as described by Maet al. (Ma et al. (2014). Pharmaceutical Research 31(9), pp. 2393-2403,incorporated by reference in its entirety for all purposes).

The coating solution was composed of 1% (w/v) carboxymethylcellulose(CMC) sodium salt (low viscosity, USP grade, CarboMer, San Diego,Calif., USA), 0.5% (w/v) Lutrol F-68 NF (BASF, Mt. Olive, N.J., USA),and fluorescent OVA labeled with fluorescein as a model allergen. CMCand Lutrol F-68 are FDA approved for injection, and are thus safeexcipients to use. Coated microneedle arrays were inserted in mouse skinfor 5 min (FIG. 5). Mouse skin was first prepared by carefully trimmingthe hair and then by applying hair-removing lotion. The mass of OVA onfresh microneedle array (M1), on microneedle array after insertion (M2),and on skin surface (obtained by using a cotton tip and extracting inwater) (M3) was quantified using fluorescent spectroscopy and a standardcurve of fluorescein-OVA. The amount of OVA delivered into skin was thenobtained (M1-M2-M3), and converted into percent delivered by dividingwith M1. Greater than 70% of OVA coated on MNs was delivered into themouse skin (FIG. 6).

Example 2—Mouse Model for Peanut Allergy

To assess the therapeutic efficacy of peanut extract (PE) coatedmicroneedles, a mouse peanut allergy model was established. Using apreviously published protocol (Dioszeghy et al. (2014). Clin. Exp.Allergy 44(6): pp. 867-881, incorporated by reference herein in itsentirety for all purposes) mice were sensitized to peanut by oral gavagewith 1 mg PE+10 μg cholera toxin (CT) weekly for six weeks (FIG. 7). Tocheck if mice were successfully made allergic to PE, the mice werechallenged orally with 20 mg PE (10 mg+10 mg at 30 min interval), andbody temperature and clinical scores were recorded. Significant drop(p<0.0001) in body temperature (FIG. 8, left) and significantly higheranaphylactic score (FIG. 8, right) in sensitized mice in comparison tocontrol naïve mice verified the progression of allergic reaction insensitized mice. Five minutes post challenge, blood was also collectedto analyze anti-PE IgE antibodies and histamine, which is released bymast cells and basophils during an allergic reaction. An elevated levelof anti-PE IgE (FIG. 9, left) and histamine (FIG. 9, right) furtherverified successful development of the mouse peanut allergy model.

Example 3—Generation of Peanut Extract Specific Antibodies

To determine the ability of peanut extract (PE) coated microneedles togenerate an immune response, microneedles coated with 1, 5 or 25 μg PEwere used to immunize naïve mice three times (one dose per week) (FIG.10). Six weeks later (at day 56), mice were bled to check for anti-PEresponses. The mice were then euthanized and their bone marrows andspleens were aseptically collected. All three PE doses were able toinduce PE-specific IgG, IgG1 and IgG2a antibodies (FIG. 11). The 5 μgand 25 μg PE doses had similar antibody levels, while the 1 μg PE doseinduced slightly lower anti-PE antibodies, although the difference wasnot statistically significant (p>0.05).

The antibody response from peripheral B cells of the bone marrow cellswas also evaluated. All IgG subtypes and IgE were detectable insupernatant of bone marrow cultures irrespective of the dose (FIG. 12).Low anti-P E IgE responses are indicative that microneedle-basedallergen immunotherapy does not cause sensitization to peanut. Withoutwishing to be bound by theory, it is thought that the ability to detectantibody secretion in the bone marrow offers the possibility thatlong-term plasma cells that secrete anti-PE antibodies might bestimulated, which might imply the ability to generate long termsustained unresponsiveness to peanut allergen through microneedle basedpeanut immunotherapy.

Example 4—Assessment of Immune Response

To assess the nature of immune response (Th1 vs Th2) induced byPE-coated microneedles, splenocytes (from spleens as collected inExample 3, above) were cultured in vitro, and re-stimulated with PE (200μg/ml). After 72 hr. of re-stimulation, supernatants were collected toanalyze the secreted cytokines. Both Th1 (IL-2 & IFN-7) and Th2cytokines (IL-4 & IL-5) were secreted irrespective of PE dose.Expression of IL-2 was higher in 5 μg PE group than the 25 μg PE, whileIFN-7 was observed higher in 1 μg PE group (FIG. 13). There was noconsiderable difference observed in IL-4 and IL-5 expression between the1, 5, and 25 μg PE doses. Microneedles thus appear to induce a mixedTh1/Th2 response.

Example 5—Assessment of Anaphylactic Shock in Peanut Sensitized Mice

In this experiment, it was determined whether peanut extract (PE) coatedmicroneedles provide a therapeutic effect in peanut sensitized(allergic) mice. The experimental protocol is shown in FIG. 14. First,mice were sensitized to peanut as described above in Example 2. Then,after a rest of three weeks, the microneedle cutaneous immunotherapy(CIT) group received g PE coated on microneedles every week for a totalof three weeks. After a rest of three more weeks, mice were challengedorally with a high dose of PE (20 mg/mouse: 10 mg+10 mg delivered at 30min interval via oral gavage) (FIG. 14).

The following control groups were included: (i) peanut sensitized micethat did not receive microneedle-CIT treatment but received oral PEchallenge, (ii) naïve mice that received oral PE challenge, and (iii)naïve mice without treatment or oral challenge. For all groups, fiveminutes after oral PE challenge, mice were bled to collect plasma foranalysis of inflammatory markers. Mice were monitored every 10 min. toassess the severity of anaphylaxis based on a scoring system describedpreviously (see, McCaskill et al. (1984). Immunology 51(4), pp. 669-77,incorporated herein by reference in its entirety for all purposes), andfor change in body temperature measured with a rectal probe. Themicroneedle-CIT group had higher anti-PE IgG, IgG1 and IgG2a in plasmaas compared to the allergic/sensitized but untreated group (FIGS. 15 and16). Anti-PE IgE levels were significantly lower in the microneedle-CITgroup as compared to the untreated group. Moreover, lower score ofanaphylaxis, and low expression of histamine and mast cell protease-1(MCPT-1) in plasma of mice that were treated with microneedle-CIT ascompared to untreated group further demonstrated the therapeuticefficacy of microneedle-CIT (FIG. 17). No considerable differences wereobserved in change of body temperature between the different groups.

Example 6—Peanut Allergen Dose Titration

Naïve mice will be given 0.1 μg, 0.3 μg, 0.6 g, 1 μg, 2 g, or 5 μgpeanut allergen, in the form of peanut extract (PE) coated onmicroneedles, once a week for 12 weeks. Blood will be collected everytwo weeks to measure anti-PE antibodies set forth in FIG. 11 and thecorresponding Example. At the end of the 12-week period, mice will beeuthanized, and bone marrow and spleen will be collected to analyzeantibody secreting cells in bone marrow, and cytokines from splenocyterestimulation (e.g., the antibodies set forth in FIG. 11 and thecytokines set forth in FIG. 13, and the corresponding Examples). Sham(microneedles coated with excipients but no PE) and naïve groups will beincluded as controls.

Example 7—Comparison of Microneedle Lengths

The immune response generated from microneedles (MNs) of various lengths(e.g., 200 μm, 300 μm, 400, μm, 500 μm, 600 μm and 700 μm will beassessed.

The delivery efficiency from MNs of different lengths will be evaluatedas by coating MNs with fluorescent Ova and measuring the fluorescencedelivered via the microneedles (see, e.g., FIG. 6).

Immune response will be characterized by measuring the antibodies and/orcytokines described previously in FIGS. 11 and 13 and the correspondingExamples.

Example 8—Comparison of Allergen Delivery with and without Adjuvant

The effect of cGMP and cAMP, which are ligands of Stimulator ofInterferon Genes (STING) (also known as transmembrane protein 173(TMEM173)) was evaluated (FIG. 18). cGMP and cAMP were added to coatedmicroneedle formulation containing ovalbumin as a model allergen. FIG.18 shows an increased Th1 response (higher IgG2a) for the compositionscontaining the STING ligands, as compared to ovalbumin compositionalone.

The effect of CpG as an adjuvant was evaluated on IgG response (FIG.19). CpG (#1826, a mouse specific CpG, 5′-tccatgacgttcctgacgtt-3′: 20nucleotides with bases having phosphorothioate bonds to make it nucleaseresistant) was added to microneedle coating compositions containingovalbumin as a model allergen. Addition of CpG adjuvant increased totalIgG and IgG2a (Th1 type response) as compared to ovalbumin without CpGas adjuvant (FIG. 19).

The effect of CpG was assessed with peanut extract as the allergen.Results are shown in FIG. 20. When CpG (25 μg) was included incompositions containing 25 μg peanut extract and coated on microneedles,there was some increase in total IgG and IgG2a as compared tocompositions containing PE without CpG. Further, CpG significantlyreduced anti-peanut IgE levels (FIG. 20).

Addition of adjuvants in MN coating compositions for the treatment ofpeanut allergy was tested. The schedule set forth in FIG. 21 wasfollowed. Mice were sensitized to peanut. Allergic mice were treatedwith either peanut extract (PE) or peanut extract+CpG (PE+CpG), andsubsequently, mice were orally challenged with peanut to test thetreatment efficacy. None of the mice in PE+CpG group (0/8) had ananaphylactic score of greater than 2 while in the PE group (no CpGusage) 3/8 mice had a score >3 (FIG. 22). The anaphylactic score is ameasure of severity of the anaphylactic reaction with lower scoreindicating a less severe reaction.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. In embodiments of any of the compositions andmethods provided herein, “comprising” may be replaced with “consistingessentially of” or “consisting of”. As used herein, the phrase“consisting essentially of” requires the specified integer(s) or stepsas well as those that do not materially affect the character or functionof the claimed invention. As used herein, the term “consisting” is usedto indicate the presence of the recited integer (e.g., a feature, anelement, a characteristic, a property, a method/process step or alimitation) or group of integers (e.g., feature(s), element(s),characteristic(s), property(ies), method/process steps or limitation(s))only.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, words of approximation such as, without limitation,“about”, “substantial” or “substantially” refers to a condition thatwhen so modified is understood to not necessarily be absolute or perfectbut would be considered close enough to those of ordinary skill in theart to warrant designating the condition as being present. The extent towhich the description may vary will depend on how great a change can beinstituted and still have one of ordinary skill in the art recognize themodified feature as still having the required characteristics andcapabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by aword of approximation such as “about” may vary from the stated value byat least 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

To aid the Patent Office, and any readers of any patent issued on thisapplication in interpreting the claims appended hereto, applicants wishto note that they do not intend any of the appended claims to invokeparagraph 6 of 35 U.S.C. § 112 as it exists on the date of filing hereofunless the words “means for” or “step for” are explicitly used in theparticular claim.

For each of the claims, each dependent claim can depend both from theindependent claim and from each of the prior dependent claims for eachand every claim so long as the prior claim provides a proper antecedentbasis for a claim term or element.

1.-166. (canceled)
 167. A method for treating a peanut allergy in asubject sensitized to the peanut allergen and in need thereof,comprising, delivering an effective amount of the peanut allergen intothe subject's cutis skin layer, wherein the delivering step is carriedout one or more times during an administration period, and comprises,(i) inserting one or more solid microneedles each comprising a base,shaft and tip into the subject's skin, wherein at least one microneedleof the one or more solid microneedles does not extend beyond the cutisonce inserted, and is coated with an amount of the allergen; and (ii)allowing the peanut allergen to dissociate from the at least onemicroneedle while inserted in the subject's cutis; and (iii) removingthe one or more solid microneedles from the subject's skin.
 168. Themethod of claim 167, wherein the one or more solid microneedles extendsfrom an adhesive substrate.
 169. The method of claim 167, wherein theone or more solid microneedles is stainless steel.
 170. The method ofclaim 167, wherein the one or more solid microneedles is present in amicroneedle array extending from a common substrate.
 171. The method ofclaim 167, wherein the average width of the one or more microneedles, asmeasured at the widest cross section of each respective microneedle ofthe one or more microneedles, is from about 20 μm to about 500 μm, orfrom about 50 μm to about 350 μm, or from about 100 μm to about 250 μm.172. The method of claim 167, wherein the one or more microneedlescomprises a plurality of microneedles, and substantially all themicroneedles of the plurality are coated with the allergen.
 173. Themethod of claim 167, wherein the one or more microneedles comprises fromabout 10 to about 200 microneedles.
 174. The method of claim 167,wherein the peanut allergen comprises Ara h1, Ara h2, Ara h3, Ara h4,Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Arah13, a peptide fragment thereof, or a combination thereof.
 175. Themethod of claim 167, wherein the one or more solid microneedles iscoated with an adjuvant.
 176. The method of claim 167, wherein eachmicroneedle of the one or more microneedles does not extend beyond theepidermis skin layer.
 177. The method of claim 167, wherein eachmicroneedle of the one or more microneedles does not extend beyond thedermis skin layer.
 178. The method of claim 167, wherein at least about40% of the allergen disassociates from the at least one microneedlewhile inserted in the subject's cutis.
 179. The method of claim 167,wherein the peanut allergen dissociates from the at least onemicroneedle for about 1 minute to about 10 minutes.
 180. The method ofclaim 167, wherein the delivering is carried out once daily during theadministration period.
 181. The method of claim 167, wherein thedelivering an effective amount of an allergen results in delivering anescalating dosage of the allergen at least once during theadministration period.
 182. The method of claim 167, wherein thedelivering an effective amount of an allergen results in delivering anescalating dosage of the allergen at least twice during theadministration period.
 183. The method of claim 167, wherein thedelivering an effective amount of an allergen results in delivering anescalating dosage of the allergen at least three times during theadministration period.
 184. The method of claim 167, wherein thetreating comprises in desensitization to the allergen.
 185. The methodof claim 167, wherein treating comprises decreasing the number of peanutallergen specific IgE antibodies in the subject, as compared to thenumber of peanut allergen specific IgE antibodies secreted by thesubject prior to the treating.
 186. The method of claim 167, whereintreating comprises increasing the number of peanut allergen specific IgGantibodies in the subject, as compared to the number of peanut allergenspecific IgE antibodies secreted prior to the treating.